Compositions and methods for treatment of neurological disorders and neurodegenerative diseases

ABSTRACT

It has been discovered that the stimulation of β-adrenergic receptors, which activate cAMP formation, give rise to increased APP and GFAP synthesis in astrocytes. Hence, the in vitro or in vivo exposure of neuronal cells to certain compositions comprising β-adrenergic receptor ligands or agonists, including, e.g., norepinephrine, isoproterenol and the like, increases APP mRNA transcription and consequent APP overproduction. These increases are blocked by β-adrenergic receptor antagonists, such as propranolol. The in vitro or in vivo treatment of these cells with 8Br-cAMP, prostaglandin E 2  (PG E 2 ), forskolin, and nicotine ditartrate also increased APP synthesis, including an increase in mRNA and holoprotein levels, as well as an increase in the expression of glial fibrillary acidic protein (GFAP). Compositions and methods are disclosed of regulating APP overexpression and mediating reactive astrogliosis through cAMP signaling or the activation of β-adrenergic receptors. It has further been found that the increase in APP synthesis caused by 8Br-cAMP, PG E 2 , or forskolin is inhibited by immunosuppressants, immunophilin ligands, or anti-inflammatory agents, such as cyclosporin A, and FK-506 (tacrolimus), as well as ion-channel modulators, including ion chelating agents such as EGTA, or calcium/calmodulin kinase inhibitors, such as KN93. The present invention has broad implications in the alleviation, treatment, or prevention of neurological disorders and neurodegenerative diseases, including Alzheimer&#39;s Disease.

RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of co-pendingU.S. application Ser. No. 08/924,505 filed Sep. 5, 1997, which isrelated to U.S. Provisional Application No. 60/025,507, filed Sep. 5,1996, and U.S. Provisional Application No. 60/033,765, filed Jan. 15,1997.

STATEMENT OF FEDERAL SUPPORT

[0002] The present invention is made in whole or in part with financialsupport from the Federal Government under grant NIH #MH-28783. TheFederal Government may have rights in the invention.

1. FIELD OF THE INVENTION

[0003] The present invention relates to compositions and methods for thetreatment of various neurological diseases and neurodegenerativedisorders, particularly those affected by an overabundance of AmyloidPrecursor Protein (APP). In particular, it has been discovered that APPsynthesis is stimulated by activation of cell surface receptors coupledto the formation of cyclic adenosine monophosphate (cAMP). Moreover, ithas been found that certain substances can inhibit APP synthesis, eitherdirectly or by antagonizing receptors coupled to cAMP formation.

2. BACKGROUND OF THE INVENTION

[0004] Alzheimer's Disease (AD) is the most common neurodegenerativedisorder of aging, and is characterized by progressive dementia andpersonality dysfunction. The abnormal accumulation of amyloid plaques inthe vicinity of degenerating neurons and reactive astrocytes is apathological characteristic of AD.

[0005] As the fourth leading cause of death in industrialized societies,surpassed only by heart disease, stroke and cancer, AD affects 5-11% ofthe population over the age of 65 and 30% of those over the age of 85.The estimated cost of caring for the approximate 2.5-4.0 million ADcases in the USA exceeded $60 billion in 1991 alone. Considering theestimated 17-25 million existing AD cases worldwide, AD will no doubtbecome an escalating healthcare problem of unparalleled proportions asthe world's geriatric population grows. Much work remains in the questto find an effective treatment for AD.

[0006] APP processing is regulated by neurotransmitters and synapticactivity. Amyloid plaques in AD accumulate near dystrophic neurons andreactive astrocytes. B. Cordell, Annu. Rev. Pharmacol. Toxicol. 34, 69(1994); D. J. Selkoe, Annu. Rev. Neurosci. 17, 489 (1994). Theactivation of neurotransmitter receptors, which are coupled tophosphotidylinositol (PI) hydrolysis or to protein kinase C (PKC)activation, can promote APP metabolism and decrease amyloid formation.R. M. Nitsch, B. E. Slack, R. J. Wurtman, J. H. Growdon, Science 258,304 (1992); B. A. Wolf et al., J. Biol. Chem. 270, 4916 (1995); J. D.Buxbaum, A. A. Ruefli, C. A. Parker, A. M. Cypess, P. Greengard, Proc.Natl. Acad. Sci. U.S.A. 91, 4489 (1994); R. K. K. Lee, R. J. Wurtman, A.J. Cox, R. M. Nitsch, Ibid., 92, 8083 (1995); Ulus and Wurtman,J.Pharm.Exp.Ther., 281,149 (1997); Lee et al., PNAS USA, 92, 8083(1995). Activation of neurotransmitters coupled to cAMP productionsuppresses both constitutive and PKC/PI-stimulated APPs secretion inastroglioma cells and in primary astrocytes. Eftimiopoulos et al.,J.Neurochem., 67, 872 (1996); Lee et al., J.Neurochem., 68,1830 (1997).Cyclic AMP and PK activators also increase A production, Marambaud etal., Br. J. Pharmacol. 126, 1186 (1999). The drastic alterations inneurotransmitter levels and second messenger signalling created byneurodegeneration and synapse loss in AD may disrupt APP processing inways that promote the accumulation of amyloidogenic or neurotoxic APPfragments. In contrast, the loss of various neurotransmitters in AD mayincrease cellular levels of APP holoprotein containing amyloidogenic orneurotoxic peptides due to a decrease in proper APP metabolism. B. A.Yankner et al., Science, 245, 417 (1989); M. R. Kozlowski, A.Spanoyannnis, S. P. Manly, S. A. Fidel, R. L. Neve, J. Neurosci. 12,1679 (1992).

[0007] Increased APP production in Down's syndrome/Trisomy 21 isassociated with a high incidence of AD at an early age due to the extracopy of the APP gene. Overexpression of APP in cell cultures and intransgenic mice is also associated with neurodegeneration and withage-related cognitive deficits, suggesting that overexpression of APPcould contribute to the neuropathology of AD. K. Maruyama, K. Terakado,M. Usami, K. Yoshikawa, Nature, 347, 566 (1990); K. K. Hsiao et al.,Neuron 15, 1203-1218 (1995); P. M. Moran, L. S. Higgins, B. Cordell, P.C. Moser, Proc. Natl. Acad. Sci. U.S.A. 92, 5341 (1995).

[0008] Several APP isoforms, ranging in size from 695-770 amino acids,are derived by differential splicing of a primary transcript. Of thethree major APP isoforms, APP695 is predominantly expressed in neurons;APP751 and APP770, which harbor an additional Kunitz-type proteaseinhibitor (KPI) insert at the N-terminus, are predominantly expressed inastrocytes and appear to be increased in AD brain. T. E. Golde, S.Estes, M. Usiak, L. H. Younkin, S. G. Younkin, Neuron 4, 253 (1990); R.L. Neve, E. A. Finch, L. R. Dawes, Ibid., 1, 669 (1990); J. P. Andersonet al., EMBO J. 8, 3627 (1989); C. Nordstedt et al., Proc. Natl. Acad.Sci. U.S.A. 88, 8910 (1991). The decreased amounts of APP695 inpostmortem AD brains may be due to neuronal loss. The increase inKPI-containing APP isoforms in AD and in regions surrounding senileplaques raises the possibility that transcriptional activation of APPsynthesis in astrocytes contributes to AD neuropathology.

[0009] 2.1. Prior AD Studies

[0010] Aging, neurodegeneration and synapse loss in AD are associatedwith astrocyte proliferation and an upregulation of KPI-containing APPisoforms. See, e.g., A. Brun, X. Liu, C. Erikson, Neurodegeneration 4,171 (1995); R. Schechter, S. H. C. Yen, R. D. Terry, J. Neuropathol.Exp. Neurol. 40, 95 (1981); L. A. Hansen, D. N. Armstrong, R. D. Terry,Neurobiol. Aging 8, 1 (1987); K. Iverfeldt, S. I. Walaas, P. Greengard,Proc. Natl. Acad. Sci. U.S.A. 90, 4146 (1993).

[0011] McGeer, P. L. et al., in The Lancet, 335, 1037 (1990), presentthe results of a retrospective study that revealed an apparently lowincidence of Alzheimer's Disease in rheumatoid arthritis patients. Theseauthors propose the possibility that anti-inflammatory therapy conferssome protection against AD. While provocative, the authors' proposal isbased solely on circumstantial evidence. This fact is not lost on theauthors, who note three alternative explanations for their observations,in addition to the possible protective role of anti-inflammatorytherapy.

[0012] Andersen, K. et al., in Neurology (August 1995) 45:1441, describethe results of their retrospective study. This article, perhaps,illustrates the care that one should take in conducting studies “inhindsight” because of the danger of over-interpretation orover-manipulation of the data in an effort to enhance any perceiveddifferences. To their credit, the authors tempered their conclusions,stating that their findings are “compatible” with a possible protectingeffect of NSAIDs (non-steroidal anti-inflammatory drugs) on the risk ofAD. The authors fairly point out that important issues remain, includingwhether the presence of complement leads to neurodegeneration or whetherthe activation of complement is brought about by the cell's need tophagocytose damaged neurons, how long one has to be exposed to NSAIDs toobtain clinically detectable results, and the need for studies that arebetter designed. The article adds that no relationship between NSAIDsexposure and cognitive function is found.

[0013] In contrast, an earlier article by Rich, J. B. et al., whichappeared in Neurology (January 1995) 45:51, reported on the results oftheir review of the records of 210 Alzheimer's patients. These authorsconcluded that patients on NSAIDs performed better on certain tests,including Mini-Mental State Examination, Boston Naming Test, delayedBenton Visual Retention Test, among others, versus non-NSAID patients.However, no significant difference is found in an even greater number ofother tests performed, including Block Design, Immediate Benton VisualRetention Test, Gollin Incomplete Figures Test, to name a few.Recognizing the inherent limitations of their study, the authors statethat “[m]ethodologic limitations inherent in retrospective studies suchas this one preclude us from addressing the specificity of theprotective effects of NSAIDs.” Indeed, the patients examined are likelyto be on several types of medication at once.

[0014] In fact, in an earlier study by Lindsay, J. and co-workersreported in Neurology (November 1994) 44:2073, it is found that thosewith arthritis had a significantly reduced risk of Alzheimer's disease.It is also found that the use of NSAIDs gave rise statistically to alower risk. However, it is suggested that the presence of arthritisitself is the determinant in lowering the apparent risk for developingAlzheimer's disease and not the taking of NSAIDs.

[0015] One has to go back even earlier to a study by Rogers, J. et al.,in Neurology (August 1993) 43:1609, to find a controlled 6-monthinvestigation involving the administration of 100-150 mg indomethacin(an NSAID) or placebo to mild or moderately impaired Alzheimer's diseasepatients. These authors report that, based on a battery of cognitivetests, the indomethacin treatment appeared to protect those patientsreceiving indomethacin from the degree of cognitive decline exhibited bypatients receiving placebo. If anything, this article, or any that havefollowed this article, suggests that the administration of indomethacinreduces the onset of dementia in Alzheimer's patients. Never has it beendisclosed or suggested that the administration of indomethacin preventsthe overproduction of APP.

[0016] Astrocytes upregulate expression of glial fibrillary acidicprotein (GFAP) as they transform from a resting state intoprocess-bearing reactive astrocytes during aging and in brain injury.Eddleston and Mucke, Neurosci., 54, 15 (1993). GFAP levels are elevatedin brain tissue and cerebrospinal fluid in AD [Wallin et al., Dementia,7, 267 (1996)], suggesting that reactive astrocytes may contribute tothe neuropathology. Furthermore, persistent and rapid elevations in APPimmunoreactivity have been observed in GFAP-positive astrocytesfollowing brain injury. Siman et al., J.Neurosci., 3, 275 (1989); Banatiand Kreutzberg, J. Cereb. Blood Flow Metab., 12, 257 (1995). In the ADbrain , the loss of synapses is associated with an increase in thenumber of GFAP-positive astrocytes [Brun et al., Neurodegeneration, 4,171 (1995)], and increases in KPI-containing APP mRNA in the frontalcortex have also been attributed to the astrocytic response duringneuronal damage [Golde et al., Neuron, 4, 253 (1990)]. It seems that theloss of synapses and neurons in AD might initiate a pathological cascadethat includes APP synthesis by reactive astrocytes.

[0017] Cytosolic phospholipase A₂, which releases arachidonic acid fromcellular phospholipids, is elevated in AD brain and after transientglobal ischemia. Stephenson et al., Neurobiol. Disease, 3, 51 (1996);Clemens et al., Stroke, 27, 527 (1996). The cyclooxygenation ofarachidonic acid, catalyzed by two forms of cyclo-oxygenase (COX), COX-1and COX-2, produces prostaglandins which, in turn, regulateneurotransmission, immune and inflammatory responses by activatingreceptors coupled to cAMP formation. Goetzl et al., FASEB J., 9, 1051(1995). We have discovered that cAMP elevations caused by activation ofneurotransmitter receptors increased APP mRNA and holoprotein productionin astrocytes. Lee et al., PNAS USA, 94, 5422 (1997). As discussedherein, it is now shown that activation of prostaglandin E₂ (PG E₂)receptors coupled to increased cAMP formation also stimulates thesynthesis of APP mRNA and holoprotein. This effect appears to bemediated by cAMP-dependent protein kinases, and can be inhibited byvarious substances, including immunosuppressants, immunophilin ligands,and ion-channel modulators. Portions of this work have been presented asan abstract. Lee et al., J.Neurochem. (supp), 69, S103B (1997).

[0018] Epidemiologic and clinical data suggest that the usenon-steroidal anti-inflammatory drugs (NSAIDs) delays the onset of ADand reduces the progression of pathologic symptoms in Alzheimer'sdisease. McGeer and McGeer, Brain Res. Rev. 21, 195 (1995). Aspirin,like most NSAIDs, prevent inflammation and pain by inhibiting both COX-1and COX-2 enzymes. Resveratrol, a phenolic antioxidant and COX inhibitorfound in grapes, inhibits prostaglandin production, and has anti-cancerand anti-inflammatory properties. Jang et al., Science 275, 218 (1997).

[0019] Most traditional NSAIDs are more potent against the COX-1 formthan against the COX-2 form of cyclo-oxygenase. COX-1 is constitutivelyexpressed in most tissues; regulates the production of essentialprostaglandins in kidney and gastric mucosa; and is essential forplatelet thromboxane production. In contrast, COX-2 is rapidly induciblein response to cytokines, inflammation, and injury. The longterm use ofnon-specific COX inhibitors such as aspirin and related NSAIDs ispotentially toxic to the kidney, and can cause ulcers andgastrointestinal bleeding. Allison et al., N. Engl. J. Med. 327, 749(1992); Wallace Can. J. Physiol. Pharmacol. 72, 1493 (1994). COX-2 mRNAis elevated in the post-mortem brains of those with AD. Pasinetti &Aisen, Neuroscience, 87, 319-324.

[0020] Highly selective COX-2 inhibitors such as DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone)and DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone)are orally bioactive drugs. Riendeau et al., Br. J. Pharmacol. 121, 105(1997). These drugs prevent the conversion of arachidonic acid toprostaglandins.

[0021] In U.S. Pat. No. 5,385,915, Buxbaum et al. describe methods andcompositions for affecting APP processing by the administration ofagents that regulate protein phosphorylation, namely agents thatregulate kinases or phosphatases. The modulation of APP processingleads, in turn, to the regulation of the production of β/A₄ peptide, apeptide that accumulates in amyloidogenic plaques. See, e.g., col. 6,lines 8-10. Hence, Buxbaum et al. teach that one's objective shouldfocus on the search for agents that alter the metabolism of APP. Theymake no mention, teaching, or suggestion that the step preceding theprocessing of APP, that is, the expression, production, or formation ofAPP, itself, can be at all affected by select groups of substances.Indeed, as Buxbaum et al. state (at col. 21, lines 7-9), “the effectsobserved are attributable to changes in APP metabolism rather than APPtranscription” (emphasis added). Consistent with this notion, the claimsof Buxbaum et al. are drawn to a method of regulating phosphorylation ofproteins that control the processing of APP.

[0022] Similarly, in U.S. Pat. No. 5,242,932, Gandy et al. disclose andclaim a method of modulating or affecting the intracellular traffickingand processing of APP in the mammalian cell.

[0023] For additional background information on the processing of APP,release of APP derivatives, or the processing, degradation and secretionof β/A₄ APP, the interested reader is referred to the followingpublications: Nitsch, R. M. et al. Science (1992) 258:304; Lee, R. K. K.et al. Proc. Nat'l. Acad. Sci. USA (1995) 92:8083; Caporaso, G. L. etal. Proc. Nat'l. Acad. Sci. USA (1992) 89:3055; Caporaso, G. L. et al.Proc. Nat'l. Acad. Sci. USA (1992) 89:2252; and Buxbaum, J. D. et al.Proc. Nat'l. Acad. Sci. USA (1992) 89:10075.

[0024] Accordingly, it is an object of the present invention to providemethods and compositions that modulate or regulate the production orformation of APP in patients, including the expression of APP geneproducts and the transcription or translation of the APP gene in braincells. For example, the production of APP by mammalian cells, inparticular, by cells in the brain, can be increased or reduced.

[0025] In attaining this objective, it is also an objective of theinvention to inhibit excessive amyloid formation, prevent neuritedystrophy and alleviate pathological symptoms, such as neurodegenerationor cognitive deficits that may arise from the negative effects ofinappropriately expressed, produced, or formed amounts of APP.

[0026] Furthermore, the present invention seeks to provide compositionsand methods that alleviate the detrimental effects of inappropriate APPproduction arising from overstimulation of receptors, particularly thosecoupled to cAMP formation.

[0027] It is also an object of the invention to provide relief from thedebilitating effects of injury or trauma to the brain, as well asneurological diseases and neurodegenerative disorders, such asAlzheimer's, Parkinson's, or Lou Gehrig's Disease (amyotrophic lateralsclerosis), multiple sclerosis and the like, which may have their rootsin the formation or presence of amyloid plaques.

3. SUMMARY OF THE INVENTION

[0028] It has now been discovered that prolonged activation of receptorsthat are coupled to increased cAMP formation in cortical astrocytesupregulates both APP mRNA levels and APP holoprotein bearing the KPIinsert. It has further been discovered that such overproduction, whichit is shown can result from the body's response to brain injury ortrauma, can be inhibited by certain substances, including agents thatinhibit an inflammatory response and immune system suppressants orimmunophilin ligands.

[0029] In particular, it has been discovered that the immune orinflammatory response of astrocytes to brain injury accelerates theformation of amyloid plaques. It has also been shown that cAMP signalingregulates the astrocytic response to neuronal injury, while alsostimulating transcriptional activation of the APP gene. Further, it isshown that prostaglandins, which are coupled to cAMP, also stimulate APPoverexpression.

[0030] What is more, it has been shown that certain substances,including immune system suppressants (agents that inhibit aninflammatory response, and the like) or immunophilin ligands inhibitthis abnormal increase in APP synthesis (mRNA and cell-associatedprotein), which is caused by elevations in cAMP levels. Thus, thesesubstances can be used to prevent APP over-expression in brain cells.

[0031] It has further been discovered that ion-gated channels canregulate APP overexpression. This is based on the observations,discussed herein, that the activation of nicotinic receptors in culturedastrocytes increases cellular levels of APP, as well as the observationthat ion-channel modulators, such as calcium channel blockers andcalcium/calmodulin kinase inhibitors, partially inhibit the stimulatoryeffect of prostaglandins on APP synthesis.

[0032] It has further been discovered that non-steroidalantiinflammatory agents (NSAIDS), such as inhibitors of cyclo-oxygenaseactivity including, but not limited to, aspirin also prevent APPoverexpression and the overproduction of amyloidogenic peptides.

[0033] It has still further been discovered that non-steroidalantiinflammatory agents (NSAIDS), such as specific inhibitors ofcyclo-oxygenase type 2 activity including, but not limited to, DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),and resveratrol, can also prevent APP overexpression and theoverproduction of amyloidogenic peptides.

[0034] Furthermore, it has been discovered that NSAID cyclo-oxygenasetype 2 inhibitors also increase secretion of soluble APP, and reduceintracellular APP holoprotein.

[0035] The present invention also contemplates and provides an assay foridentifying or screening potential drugs that can inhibit the excessiveor inappropriate production of amyloidogenic or neurotoxic APPfragments. For example, such an assay may utilize a tissue or cellculture comprising brain cells, as described herein.

[0036] The invention also contemplates a method by which theoverexpression of APP is deliberately effected, followed by thepromotion or stimulation of APP metabolism to provide soluble APP(APPs). The former step can be attained by, for example, cAMP signaling,while the latter process can be accomplished, for example, by theactivation of protein kinase C (PKC) or of neurotransmitter agonists(e.g., via m1, m3, serotoninergic, or metabotropic glutamate receptors)which increase phophotidylinositol (PI) hydrolysis. Consequently,increased amounts of APPs are secreted into the medium, and theformation of amyloidogenic Aβ peptides is disrupted. It is believed thatsecreted APPs have neurotrophic and neuroprotective functions. SecretedAPPs have been shown to promote neurite outgrowth and maintain synapse.It is believed further that increased APPs secretion promotes synaptictransmission and neuronal regeneration (e.g., via neurite or axonaloutgrowth). The net result is the conversion of an amyloidogenic event(i.e., APP overexpression and Aβ formation) into a neurotrophic event(i.e., APPs secretion).

[0037] The invention further contemplates that decreasing overexpressionof amyloidogenic APP and amyloidogenic APP peptides is beneficial intreatment of subjects with neurodegenerative conditions, includingimmune or inflammatory conditions associated with Alzheimers Disease,stroke, cerebral ischemia, de-myelinating conditions, and mechanicalinjury. Decreasing overexpression of amyloidogenic APP and amyloidogenicAPP peptides is also contemplated in the present invention as beneficialin the prevention of neurodegenerative diseases including AlzheimersDisease.

[0038] These and other objects of the invention will be evident to thoseof ordinary skill from a consideration of the discussions anddescriptions provided in this specification, including the detaileddescription of the preferred embodiments.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0039]FIG. 1A illustrates the effects of norepinephrine (NE) andpropranolol on the expression of cell-associated APP (APP holoprotein);FIGS. 1B and 1C show that norepinephrine or the β-adrenergic agonistisoproterenol stimulates APP mRNA production, and that these stimulatoryeffects are abolished by the antagonist propranolol.

[0040]FIG. 2 illustrates the effect of propranolol on the changes in PIhydrolysis and cAMP formation caused by NE treatment.

[0041]FIGS. 3A and 3B illustrate the effects of PMA anddexnorfenfluramine (DNF) on cell-associated APP and APPs secretion.

[0042]FIGS. 4A and 4B illustrate the effects of 8Br-cAMP and forskolinon cell-associated APP and GFAP from cultured astrocytes.

[0043]FIGS. 5A and 5B illustrate the effects of 8Br-cAMP on APPssecretion from cultured astrocytes.

[0044]FIG. 6 illustrates the effect of 8Br-cAMP on APP mRNA.

[0045]FIG. 7 illustrates the effects of 8Br-cAMP or NE on actin and APPmRNA.

[0046]FIG. 8 illustrates the effects of forskolin or 8Br-cAMP on GFAPexpression in cultured astrocytes.

[0047]FIGS. 9A, 9B and 9C illustrate the effect of cyclosporin A on APPssecretion and cell-associated APP, in the absence or presence of8Br-cAMP.

[0048]FIGS. 10A, 10B, 10C and 10D illustrate the effects of PG E₂ inincreasing cellular levels of APP holoprotein, levels of secreted APP,and cellular cAMP levels.

[0049] FIGS. 11A and lab illustrate the effects of 8Br-cAMP andforskolin on cellular cAMP levels, and on levels of APP mRNA, APPholoprotein, and GFAP.

[0050]FIGS. 12A and 12B illustrate the effects of Sp-cAMPS triethylamineon APP holoprotein levels, and the effects of the PKA inhibitor H-89 onAPP mRNA and APP holoprotein stimulated by PG E₂.

[0051]FIGS. 13A, 13B and 13C illustrate the effects of cyclosporin A orFK-506 on increases in APP mRNA, APP holoprotein, and GFAP levelsstimulated by PG E2 or forskolin.

[0052]FIG. 14 illustrates the effects of cyclosporin A and FK-506 oncellular cAMP levels stimulated by PG E₂.

[0053]FIGS. 15A, 15B and 15C illustrate the effects of nicotineditartrate in increasing cellular levels of APP holoprotein, and theeffects of EGTA and KN-93 on levels of APP holoprotein stimulated by PGE₂.

[0054]FIGS. 16A and 16B illustrate the effect of aspirin or resveratrolon APP expression and secretion in primary cultured astrocytes. Aspirin(A) or resveratrol (B) treatment promotes APPs secretion and decreasesthe levels of amyloidogenic APP holoprotein in cultured astrocytes.

[0055]FIG. 17A and 17B illustrate the effect of DFU or DFP on APPexpression and secretion in primary cultured brain cells. Treatment ofcultured astroyctes with DFU (A) or of cultured neurons with DFP (B)promotes APPs secretion and decreases the levels of amyloidogenic APPholoprotein in cultured astrocytes (*, significantly different fromcontrol; p<0.05).

[0056]FIG. 18 illustrates the effect of synapse loss induced byp-chloroamphetamine (PCA) on APP holoprotein levels in rats treated withor without aspirin. PCA induced significant increases in brain APPholoprotein levels, and this increase is attenuated by aspirin. Aspirintreatment alone also caused a small, but significant decrease in brainAPP holoprotein levels (*, significantly different from control;p<0.05).

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Cultures of cortical astrocytes can be obtained from postnatalrats. See, e.g., K. D. McCarthy and J. de Vellis, J. Cell Biol. 85, 890(1980).

[0058] The inventors have shown that the activation of adrenergicreceptors present in the cortical astrocytes by 50 μM norepinephrine(NE) for a period of about 24 h increases the amount of cell-associatedAPP holoprotein by ˜1.7-fold relative to amounts in untreated cells(See, FIG. 1, A), as measured using Western blots. The stimulatoryeffect of NE on total APP protein is inhibited by the β-adrenergicantagonist propranolol (50 μM) which, on its own, has no significanteffect on basal APP protein levels.

[0059] On Northern blots, APP mRNA levels are about 1.7-fold and about1.6-fold higher after 24 h treatment with NE (50 or 100 μM) or theβ-adrenergic agonist isoproterenol (50 μM), respectively, than those inuntreated cells (FIG. 1, B); these increases are also abolished by 50 μMpropranolol (FIG. 1, C). However, astrocytic levels of APP mRNA or ofholoprotein do not increase linearly with increasing NE concentrations(50-400 μM). NE can stimulate cAMP formation and PI hydrolysis byactivating β- or α₂-adrenergic receptors, respectively. Propranolol (50μM) inhibits the NE-induced (50 μM) increase in cAMP formation but notthe increase in PI hydrolysis (FIG. 2), suggesting that activation ofβ-adrenergic receptors stimulates APP gene expression in culturedcortical astrocytes by enhancing cAMP synthesis.

[0060] Increases in β-adrenergic receptor density in the hippocampus andcortex of AD brain have been attributed by others to the proliferationof astrocytes. See, N. Kalaria et al., J. Neurochem. 53, 1772 (1989).Aberrant activation of these receptors, perhaps by circulating NE in ADor through a damaged blood-brain barrier, may cause abnormal cAMPsignaling and, thus, APP overexpression in astrocytes.

[0061] In various cell lines, exposure to phorbol ester or tointerleukin-1 can increase APP mRNA production by activating PKC and theAP-1 binding site on the APP promoter. See, D. Goldgaber et al., Proc.Natl. Acad. Sci. U.S.A., 86, 7606 (1989).

[0062] Since NE also increased PI hydrolysis, the second messengersdiacylglycerol and inositol trisphosphate generated by PI hydrolysiscould also have promoted APP expression in cortical astrocytes byactivating PKC. However, direct activation of PKC by phorbol12-myristate 13-acetate (5 μM) or activation of serotoninergic receptorsby dexnorfenfluramine (100 μM), which also stimulates PI hydrolysis by˜2.6-fold relative to untreated astrocytes (p<0.05), does not increaseAPP mRNA or holoprotein levels but does increase APPs secretion by ˜3.0-and ˜2.2-fold, respectively, to that of untreated cells (FIG. 3). Theseresults indicate that activation of PKC or of receptors coupled to PIhydrolysis does not stimulate APP synthesis in astrocytes but doespromote APP metabolism.

[0063] To confirm that increased APP synthesis is mediated by elevationsin cAMP levels caused by βadrenergic receptor stimulation, the inventorshave exposed astrocytes to the membrane-permeant 8-Bromo-cAMP (8Br-cAMP)or to the adenylate cyclase activator forskolin. The levels of APPholoprotein in astrocytes increases linearly with increasingconcentrations of 8Br-cAMP or forskolin after 24 h treatment (FIG. 4) asrevealed by the use of known monoclonal antibodies (mAb22C11) orantiserum (R37) directed against the N- or C-terminus of APP,respectively. The inventors have also shown from the use of the knownantiserum R98, which recognizes an epitope of the KPI domain, thatKPI-containing APP isoforms in cortical astrocytes are also increased bytreatments that elevate cAMP levels.

[0064] APPs levels in astrocytes that are treated with or without8Br-cAMP (250 μM) for 24 h do not differ significantly (p>0.05),suggesting that decreases in APP metabolism do not account for theincrease in astrocytic APP protein that is observed with 8Br-cAMPtreatment FIGS. 5A and 5B. Northern blot analyses show that treatmentwith 250 μM 8Br-cAMP for 24 h increases astrocytic APP mRNA levels to˜1.8-fold those of untreated cells (FIG. 6). This result indicates thatprolonged (˜24 h) cAMP signaling in cultured astrocytes can stimulateAPP synthesis by transcriptional activation. Although exposure to 50 or100 μM 8Br-cAMP occasionally increases APP mRNA, these effects are notconsistent. No significant changes in APP mRNA levels are detected after6 h or 12 h treatment with 8Br-cAMP (250 μM). The inventors have alsoobserved that the APP transcript in astrocytes treated with or without8Br-cAMP has a slower mobility than that observed in neurons. Hence, theAPP mRNA that is upregulated by 8Br-cAMP treatment may contain the KPImotif.

[0065] The increases in APP mRNA that is caused by 8Br-cAMP (250 μM) orNE (50 μM) are associated with decreases in levels of mRNA for β-actin(FIG. 7). The downregulation of cytoskeletal stress fibres, such asactin, may be related to the morphological differentiation of astrocytesfrom flat, polygonal cells to process-bearing, stellate cells. Moreover,GFAP expression in cultured astrocytes is also increased by elevationsin cAMP levels (FIG. 4; FIG. 8). These morphological and biochemicalchanges that are induced by 8Br-cAMP in cultured astrocytes resemble thegliotic response of astrocytes in vivo. These findings thus suggest thatthe increased APP immunoreactivity in astrocytes after neuronal injuryresults from increased APP gene expression rather than from theinternalization of exogenous APP that are released from dying cells. Itis noted that Gegelashvili et al. reported that treatment with dibutyrylcAMP in serum-containing medium increased APP mRNA in rat glioma cellsbut not in cortical astrocytes. See, G. Gegelashvili, E. Bock, A.Schousboe, D. Linnemann. Mol. Brain. Res. 37, 151 (1996). Apparently,the stimulatory effect of cAMP on APP synthesis in cortical astrocytesof the present invention is related to the use of serum-deprived cells.

[0066] Induction of gene transcription by cAMP can be inhibited by theimmunosuppressant cyclosporin A. In cortical astrocytes, cyclosporin A(1, 5, or 10 μM) inhibits the increase in APP mRNA and APP holoproteinwhich is caused by 24 h exposure to 8Br-cAMP (250 μM). The inhibitoryeffects of 1, 5, or 10 μM cyclosporin A do not differ significantly(FIG. 9). Basal APP holoprotein levels, APPs secretion and GFAPexpression also are unaffected by these concentrations of cyclosporin A(p>0.05). Hence, immunosuppressants such as cyclosporin A exhibit apotential usefulness for preventing aberrant APP expression in AD.

[0067] The results reported herein further show that activation of PG E₂receptors coupled to cAMP formation stimulates APP gene expression incultured cortical astrocytes. Increases in APP mRNA and holoprotein weredetected with 1, 10 or 100 μM PG E₂ treatment of astrocytes for 24 h.Shorter duration treatment (6 or 12 h) with 10 μM PG E₂ did not reliablyincrease APP synthesis (unpublished data). Because about 95% of thecells in our cultures express proteins specific for astrocytes but notfor neurons or microglia [Lee and Wurtman, J.Neurochem., 68, 1830(1997)], we suggest the increased APP mRNA and holoprotein observedafter PG E₂ treatments originates from astrocytes. Since APPoverexpression causes symptoms of AD [Cordell,Annu.Rev.Pharmacol.Toxicol., 34, 69 (1994); Yoshikawa et al., Nature,359, 64 (1992); Hsiao et al., Neuron, 15, 1203 (1995)], it seems thatincreased APP synthesis in astrocytes stimulated by PG E₂ may accelerateneuropathology, and the formation of amyloidogenic and neurotoxicpeptides.

[0068] Alternative splicing of the APP gene yields several APP isoformsof vary sizes. Kang et al., Nature, 325, 733 (1987); Oltersdorf et al.,Nature, 341, 144 (1989); van Nostrand et al., Nature, 341, 546 (1989).APP695 which lacks the KPI-motif is the major isoforms found in thebrain. Astrocytes and microglia , both of which express APP751/770isoforms containing the KPI domain, express only low levels of APP mRNAand protein in the resting state but upregulate KPI-containing APPisoforms following brain injury or neurodegeneration. Siman et al.,J.Neurosci., 3, 275 (1989); Solá et al., Mol. Brain Res., 17, 41 (1993);Banati et al., J.Cereb.Blood Flow Metab., 12, 257 (1995). Our labelledAPP cDNA probe did not distinguish between the various kinds of APPtranscripts on Northern blots. However, Western blot analyses usingantiserum R98 [Kametani et al., Biochem. Biomed. Res. Comm., 191, 392(1993)] revealed increases in KPI-containing APP isoforms following PGE₂ treatments. Increases in cellular APP holoprotein were also detectedby antisera R37 directed at the C-terminus of APP, indicating that theKPI-containing APP increased by astrocytes are full-length holoproteinsharboring intact and potentially amyloidogenic Aβ peptides. SincemAb22C11 recognizes the N-termini of both APP and APP-like proteins[Weidemann et al., Cell, 57, 115 (1989); Slunt et al., J.Biol.Chem.,269, 2637 (1994)], it is possible that PG E₂ treatment may alsostimulate transcriptional regulation of other members of the APP genefamily.

[0069] APP synthesis in astrocytes is probably mediated by the increasesin cAMP production stimulated by PG E₂ treatment.Concentration-dependent elevations in cAMP were observed after treatmentwith 1, 10 or 100 μM PG E₂ but not with 0.1 μM PG E₂. Similarly, 1, 10or 100 but not 0.1 μM PG E₂ stimulated increases in APP mRNA andholoprotein. Furthermore, the stimulatory effect of PG E₂ on APPsynthesis was also mimicked by membrane-permeant 8Br-cAMP (250 μM) or byactivating adenylate cyclase with forskolin (10, 50 or 100 μM).Elevations in cAMP activate cAMP-dependent protein kinase (PKA) which,in turn, phosphorylates proteins involved in regulating gene expression.In our study, activation of PKA by Sp-cAMP triethylamine in the absenceof PG E₂ was sufficient to stimulate increases in astrocytic APPholoprotein. Furthermore, inhibition of protein kinase A by H-89dihydrochloride blocked the stimulatory effect of PG E₂ on APP mRNAproduction. These data provide strong support for PKA in mediating thestimulatory effect of cAMP on APP synthesis.

[0070] The APP promoter contains several sequences for regulatoryelements that are responsive to cAMP signalling. Salbaum et al., EMBOJ., 7, 2807 (1988). PKA can phosphorylate cAMP response element bindingprotein (CREB) to stimulate gene expression. While there is no canonicalsequence for CREB (TGACGTCA) within the 3.7-kb region upstream of theAPP transcription start site [Salbaum et al., ibid], a consensussequence for CREB (TGACCTCA) could be responsible for initiating APPsynthesis in astrocytes. Elevations in cAMP may also induce c-fos andc-jun expression to activate APP synthesis through AP-1 recognitionsites. However, APP synthesis in NG108-15 and HepG2 cells stimulated bydibutyryl cAMP appears not to depend on the presence of AP-1 or AP-2sites. Bourbonniére et al., J.Neurochem., 68, 909 (1997); Shekarabi etal., J.Neurochem., 68, 970 (1997). Nevertheless, at least two othercAMP-responsive regions have been identified within the APP promoter ofNG-108 cells [Bourbonniére et al., ibid]. It is not known if thesecis-acting regulators are functional for regulating cAMP responsivenessin astrocytes, or if the induction of APP synthesis is mediated bytransacting elements acting through the expression of othercAMP-responsive genes.

[0071] The immunosuppressants cyclosporin A and FK-506, which are alsoimmunophilin ligands, are inhibitors of calcineurin and can suppressgene activation stimulated by cAMP. Schwaninger et al., J.Biol.Chem.,270, 8860 (1995). We previously found that cyclosporin A inhibited APPsynthesis in astrocytes treated with 8-Bromo-cAMP but we did notdetermine if cyclosporin A would inhibit APP synthesis stimulated byfirst messengers. Lee et al., PNAS USA, 94, 5422 (1997). We now showthat cyclosporin A or FK-506 completely abolished APP overexpressionstimulated by PG E₂ or by forskolin. Both cyclosporin A and FK-506 bindto intracellular immunophilin receptors that are not known to directlyaffect cAMP production. Clardy, PNAS USA, 92, 56 (1995). As expected,neither cyclosporin A nor FK-506 had any effect on basal cAMP levels incultured astrocytes, and neither drug inhibited the increase in cAMPcaused by PG E₂. Hence, the inhibitory effect of cyclosporin A or FK-506appears to lie downstream of cAMP production and possibly by interferingdirectly with gene transcription. Although cyclosporin A and FK-506 arepotent immunosuppressive drugs generally used to prevent rejection oforgan transplants and autoimmune diseases, our data suggest that boththese immunosuppressants may be useful for preventing aberrant APPoverexpression.

[0072] AD is not usually considered to be an inflammatory or immunedisease. However, increased lipid peroxidation and formation ofprostaglandins have been reported in AD. Iwamoto et al., J.Neurol., 236,80 (1989); Subbarao et al., J.Neurochem., 55, 342 (1990). In addition,all major components of the classical complement pathway appear to beassociated with AD lesions, suggesting that neuronal damage or amyloiddeposits may trigger inflammatory or immune processes and accelerateneuropathology. McGeer and McGeer, Brain Res.Rev., 21, 195 (1995).Epidemiological data provide strong circumstantial evidence thatanti-inflammatory therapies such as the use of non-steroidalanti-inflammatory drugs or dapsone may be effective in slowing theprogression of neuropathology in AD. McGeer and McGeer, ibid. Theresults presented herein suggest that antiinflammatory agents such asindomethacin or inhibitors of prostaglandin G/H synthase(cyclooxygenase), such as DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),and resveratrol, which prevent the synthesis of prostaglandins fromarachidonic acid can prevent the production of amyloid and neurotoxicAPP fragments associated with APP overexpression in astrocytes.

[0073] Aspirin, like most NSAIDs, inhibits both COX-1 and COX-2 enzymes.Cultured astrocytes or neurons are treated with aspirin or resveratrolfor 1 h (at nano- or micromolar range), resulting in an increase in thesecretion of soluble APPs (as measured by Western blot analysis). Theincrease in APPs secretion caused by aspirin or resveratrol isaccompanied by a decreased levels of cellular and amyloidogenic APPholoprotein (FIG. 16). Thus, NSAIDs stimulate non-amyloidogenic APPprocessing in vitro.

[0074] Most traditional NSAIDs are more potent against COX-1 than COX-2.Since COX-2 inhibitors can offer comparable efficacy without the risksof undesirable side-effects associated with non-specific COX inhibitors,the effect of specific COX-2 inhibitors on APPs secretion and on thelevels of APP holoprotein is examined.

[0075] Highly selective COX-2 inhibitors such as DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone)and DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone)are orally bioactive drugs that prevent the conversion of arachidonicacid to prostaglandins. Treatment of cultured astrocytes with DFU or DFP(10⁻⁹−10⁻³M) for 1 h increases the secretion of soluble APPs andconcurrently decreases the levels of cellular APP holoprotein (FIG. 17).These data indicate that specific COX-2 inhibitors can prevent theexcessive accumulation of amyloidogenic APP, and also prevent theformation of amyloid by enhancing the conversion of APP to form solubleAPPs.

[0076] NSAIDs (FIG. 16), and NSAIDs that are selective COX-2 inhibitors(FIG. 17), stimulate APPs secretion and decrease the levels ofamyloidogenic APP in cultured astrocytes and neurons. NSAIDs andselective COX-2 inhibitors, by reducing prostaglandin production, mayalso suppress cAMP formation. Decreased levels of prostaglandins, andconsequently of cAMP, caused by NSAIDs or selective COX-2 inhibitors,may be responsible for the increases in APPs and decreased levels of APPholoprotein observed in the astrocyte or neuronal cultures.

[0077] Treatment with PG E₂ induced process formation and also increasedthe levels of GFAP in our cultured astrocytes. Both these effects areprobably mediated by elevations in cAMP caused by PG E₂. The disruptionof cytoskeletal fibers associated with morphological transformation intoGFAPpositive and process-bearing cells was associated with decreasedlevels of β-actin mRNA [Lee et al., PNAS USA, 94, 5422 (1997)],suggesting that these cultured astrocytes resemble reactive astrocytes.Rapid and persistent increases in APP immunoreactivity in GFAP-positivereactive astrocytes have been observed after brain lesions or ischemia.Siman et al., J.Neurosci., 3, 275 (1989); Banati et al., (1996).Although the phagocytic activity of astrocytes or microglia can increaseAPP immunoreactivity [Paresce et al., Neuron, 17, 553 (1996)], our studysuggests that GFAP-positive astrocytes can actively upregulate APPsynthesis following brain injury.

[0078] The loss of synapses has been suggested to be an early event inthe pathology of AD, and appears to be related to the extent of reactiveastrogliosis. Brun et al., Neurodegeneration, 4, 171, (1995); Heionen etal., Neuroscience, 64, 375 (1995). The invasion and proliferation ofreactive astrocytes within these regions of degeneration may explain theincreased levels of GFAP in the brain tissue and cerebrospinal fluid ofAD. Wallin et al., (1995). Indeed, the upregulation of β-adrenergicreceptors in the frontal cortex and hippocampus of AD brains has beenattributed to the proliferation of astrocytes associated withneurodegeneration. Kalaria et al., J.Neurochem., 53, 1772 (1989).Circulating levels of norepinephrine after brain injury appear to causereactive astrogliosis and cell proliferation. Hodges-Savola et al.,Glia, 17, 52 (1996). We suggested that the aberrant activation ofβ-adrenergic receptors coupled to cAMP signalling by norepinephrinemight also stimulate APP overexpression in astrocytes. Lee et al.,(1997). These studies, together with our present finding that activationof PG E₂ receptors can stimulate APP synthesis, underscore thecontribution of receptor activation in the overproduction of APP.

[0079] APP overexpression in cultured astrocytes treated with PG E₂ wasassociated with the secretion of APP holoprotein. Although secreted APPis usually truncated at the C-terminus, antisera C8 which is directed atthe C-terminus of APP [Selkoe et al., PNAS USA, 85, 7341 (1988)]detected increased amounts of APP holoprotein (˜130 kD) in the media ofastrocytes treated with PG E₂ for 24 h. The present findings areconsistent with the observation that Chinese hamster ovary cellstransfected with full-length APP751 cDNA also secrete soluble APPholoprotein. Eftimiopoulos et al., (1996). APP holoprotein can bedetected in the cerebrospinal fluid of humans, and can be activelyreleased from secretory vescicles in response to receptor stimulation orneuronal depolarization. It is not known if secreted APP holoprotein isreinternalized for subsequent processing, or if it can be metabolized inthe extracellular space.

[0080] The increase in KPI-containing mRNAs in the frontal cortex of ADpatients that are not usually expressed in the brains [Golde et al.,Neuron, 4, 253 (1990); Tanaka et al., Biochem.Biophys.Res.Comun., 165,1406 (1989)], suggest that APP isoforms with and without KPI domainshave different functions in the nervous system. The KPI domain of APP ishighly homologous to the Kunitz-type of serine protease inhibitors, andsecreted APP isoforms containing the KPI domain has been identified asprotease nexin II. Oltersdorf et al., Nature, 341, 144 (1989); vanNostrand et al., Nature, 341, 546 (1989). Protease inhibitors expressedby reactive astrocytes can also form protease-protease inhibitorcomplexes to induce the synthesis of cytokines, acute phase protein andthe migration of neurotrophils which can further promote brain injuryand inflammation. Eddleston and Mucke, Neurosci., 54, 15 (1993).Although APP overexpression can cause neurological disorders [Yoshikawaet al., Nature, 359, 64 (1992); Cordell, Annu. Rev. Pharmacol. Toxicol.,34, 69 (1994); Hsiao, Neuron, 15, 1203, (1995)], secreted andcell-associated APP may have mitogenic, neuroprotective or neurotrophicproperties. Saitoh et al., Cell, 58, 615 (1989); Schubert et al.,Neuron, 3, 689 (1989); Mattson et al., Trends Neurosci., 16, 409 (1993).In particular, the finding that APP751 promotes neurite formation andtau expression in primary neuronal cultures [Qiu et al., J.Neurosci.,2157 (1995); Lee et al., PNAS USA, 92, 8083 (1995)] suggests thatoverexpression of astrocytic APP may have effects on neuronal growth andsurvival in the brain. Whether such neurotrophic activities cancontribute to brain regeneration or to aberrant neurite outgrowthremains to be determined.

[0081] Our findings show that PG E₂ can stimulate GFAP expression, APPsynthesis and the release of amyloidogenic APP holoprotein from culturedastrocytes. APP overexpression in DS and in transgenic mice isassociated with the pathologic symptoms of AD. To the extent thatastrocytes proliferate and upregulate APP synthesis during aging andneuronal injury, non-neuronal cells may contribute to the neuronaldysfunction and the pathology of AD. Therefore, it seems that inhibitionof prostaglandin synthesis by antiinflammatory agents or by inhibitorsof phospholipase A₂ (PLA₂) may prevent APP overexpression and itsassociated pathologies. In particular, we suggest thatimmunosuppressants such as cyclosporin A or FK-506 may be especiallyuseful in preventing transcriptional activation of APP and, possibly,the progression of AD. We further suggest that, among the non-steroidalantinflammatory agents, specific inhibitors of cyclo-oxygenase-2 may beuseful in decreasing cellular production of amyloidogenic APP andamyloidogenic APP peptides and, possibly, slowing or halting theprogression of AD.

[0082] In summary, the present studies show that stimulation ofadrenergic receptors coupled to cAMP formation in astrocytes increasesthe production of APP mRNA and APP holoprotein. It is suggested that theupregulation or aberrant activation of β₂-adrenergic receptors in brainregions that are vulnerable to damage can stimulate transcriptionalactivation of APP synthesis in astrocytes and, thereby, contribute toamyloid production. Because APP overexpression can causeneurodegeneration and cognitive dysfunction, the inventors have shownthat such substances as propranolol or immunosuppressants, e.g.,cyclosporin A, FK-506, ion channel modulators, e.g., EGTA,calcium/calmodulin kinase inhibitors, e.g., KN-95, and the like, arepromising drug candidates for the treatment of AD.

5.1. Further Aspects of the Preferred Embodiments

[0083] Thus, the present invention is directed to a method of modulatingthe expression, production, or formation of amyloid precursor protein(APP) in a subject comprising administering to the subject an effectiveamount of cyclic adenosine monophosphate (cAMP), an analog of cAMP, asubstance that is a ligand, an agonist, or an antagonist of a receptorthat is coupled to the cellular levels of cAMP or to ion channels, acompound that regulates ion channels or the nuclear actions of cAMP, ora compound that regulates the activity of protein kinase A. In specificembodiments of the invention the analog of CAMP can comprise 8Br-cAMP,the ligand can comprise norepinephrine, the agonist can compriseisoproterenol, and the antagonist can comprise propranolol.

[0084] By “nuclear actions” is meant any activity of cAMP that isexerted in the nucleus of a eukaryotic cell that ultimately gives riseto changes in the expression, production, formation, metabolism, oramount of APP in a subject, including but not limited to the regulationof the promoter of the APP gene, the modulation of transcription factorsthat affect APP expression, or the stimulation of the activity ofcAMP-dependent protein kinase.

[0085] Indeed, according to the methods of the present invention, asuitable antagonist can comprise a receptor antagonist of aneurotransmitter, a modulator of signal transduction, animmunosuppressant, an anti-inflammatory agent, or combinations thereof.In a preferred embodiment, the modulator selected can modulate theactivity of protein kinase A but does not activate protein kinase C.Still in other embodiments the modulator selected does not activatephosphate activity. However, as discussed further below, in a multistepprocess having at least two or more steps, the expression, production,or formation of APP can be modulated in an initial or first step,followed by the modulation of kinase or phosphatase activity with theobjective, for example, of processing or metabolizing overproduced APPinto nonamyloidogenic or neurotrophic metabolic products.

[0086] In the inventive method the ligand, agonist, or antagonist of areceptor can be those substances that bind or exhibit an affinity for areceptor that can comprise a β-adrenergic, serotoninergic, dopaminergic,adenosine, vassopressin intestinal peptide, pituitary adenylate cyclaseactivating peptide (PACAP), prostaglandin E2, histamine, muscarinic,nicotinic, opioid, GABA, or metabotropic glutamate receptor.

[0087] The following kinase stimulators are also suitable for use in thepresent invention, including staurosporine, auranofin,N-(6-aminohexyl)-1-naphthalen-sulfonamide hydrochloride,N-(4-aminobutyl)-2-naphthalenesulfonamide hydrochloride,N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride,N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride,1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride,N-(2-(methylamino)ethyl)-3-isoquinolinesulfonamide dihydrochloride,N-(2-aminoethyl)-5-isoquinolinesulfonamide,N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride,sphingosine and tyrphostin.

[0088] Examples of additional substances, which have been found todecrease or inhibit the APP synthesis include, but are not limited toH8, H9, cyclosporin A, FK-506, Win55212, and propranolol. The inhibitoryeffects of the latter two compounds are most evident when the basallevels of APP synthesis have been upregulated. For the stimulation ofprotein kinase A (PKA), the preferred substance can comprise Sp-cAMPStriethylamine.

[0089] In practicing the disclosed method or using the disclosedcompositions the synthesis of endogenous APP can be stimulated or,alternatively, suppressed. When APP is desirably overproduced, theoverproduction can be effected by increasing the levels of cellularcAMP. The increase in the levels of cellular CAMP can, in turn, beeffected by exposing or administering to the subject effective amountsof exogenous CAMP. Preferably, the exposure or administration isperformed for a continuous period. The continuous period may be anysuitable or practical length of time but, preferably, at least about sixhours, at least about twelve hours, at least about twenty-four hours ormore.

[0090] An increase in the levels of cellular cAMP can also be effectedby stimulating the synthesis of endogenous cAMP or by retarding thebreakdown of cAMP. In a preferred embodiment of the invention, thesynthesis of endogenous cAMP is stimulated by administering an effectiveamount of a prostaglandin, such as prostaglandin E2, and the like. Othercompounds that can be used to this end include forskolin.

[0091] When it is desirable to effect a decrease of endogenous APPproduction, cellular levels of cAMP are caused to diminish. For example,the cellular levels of CAMP can be decreased by retarding the synthesisof endogenous cAMP or by stimulating the breakdown of cAMP. Substances,such as H8, H9, propranolol, anandamide, or Win55212 can be administeredto achieve this end. Also, ion-channel modulators, e.g., calcium channelblockers, including chelating agents such as EGTA, andcalcium/calmodulin kinase inhibitors, such as KN93, can be employed.

[0092] In still another embodiment of the present invention, theproduction of APP is first enhanced, followed by the stimulation of APPprocessing or metabolism. The latter step can be accomplished byadministering an effective amount of a substance that stimulates proteinkinase C (PKC) activation, activation of phosphatases, or phosphatidylinositol (PI) hydrolysis. The objective is the conversion of anamyloidogenic event (APP overexpression) to a neurotrophic event (thesecretion of APPs or other nonamyloidogenic by-products). In particular,the stimulation of PKC, for example, can be achieved with theadministration of effective amounts of phorbol ester or diacylglycerol.

[0093] The present invention also contemplates a method of modulatingthe production of amyloid precursor protein (APP) in a subjectcomprising regulating the expression of glial fibrillary acidic protein(GFAP) in the subject. Preferably, the production of APP is enhanced bystimulating the transcription or translation of GFAP mRNA. Conversely,the production of APP is diminished by inhibiting the transcription ortranslation of GFAP mRNA.

[0094] As in the methods described above, GFAP expression can beregulated by administering to the subject an effective amount of ananti-inflammatory agent, which are preferably selected from acorticosteroid, glucocorticoid, or an admixture comprising estrogen andestradiol.

[0095] An important aspect of the present invention concerns thetreatment of a subject that has suffered an injury or trauma, especiallyto the head or brain, or of a subject that is, for some other or relatedreason, may be experiencing overstimulation of cAMP expression,production, formation (or, collectively, “synthesis”).

[0096] It is particularly advantageous to treat the subject in need byadministering an effective amount of an immune system suppressant, suchas cyclosporin A or other similar suppressant.

[0097] It is noteworthy that the present invention is also directed to amethod of determining the capacity of a drug to inhibit the expression,production, or formation of amyloid precursor protein (APP) in a cellcomprising contacting a drug with a cell culture that has been exposedto cyclic adenosine monophosphate (cAMP), an analog of cAMP, a substancethat is a ligand, an agonist, or an antagonist of a receptor that iscoupled to the cellular levels of cAMP, a compound that regulates thenuclear actions of cAMP, or a compound that regulates the activity ofprotein kinase A. The level of mRNA or holoprotein produced from thecell culture in the presence of the drug is then compared with the levelof mRNA or holoprotein produced from the cell culture in the absence ofthe drug. The cell can be any type of microbial, plant, or animal cell,so long as the cell has the capacity to express, produce, or otherwiseform APP. The cell is preferably a eukaryotic cell. More preferably, theeukaryotic cell can further be a yeast cell, insect cell, invertebrate,vertebrate, or mammalian, including animal or human.

[0098] It should be apparent that the present invention is directed to amethod of alleviating the negative effects of a neurological disorder orneurodegenerative disease stemming from the aberrant expression,production, or formation of amyloid precursor protein (APP) in asubject. In a particular embodiment, an effective amount of anantagonist of a β-adrenergic receptor, which is coupled to the cellularlevels of cAMP, is administered to the subject suffering from thedisorder or disease. As described herein, the antagonist may comprise areceptor antagonist of a neurotransmitter, a modulator of signaltransduction, an immunosuppressant, an anti-inflammatory agent, orcombinations thereof, preferably provided that the modulator does notactivate protein kinase C.

[0099] It should also be apparent the present method of modulatingamyloid precursor protein (APP) expression in a subject may alsocomprise administering to the subject an effective amount of a substancethat regulates APP promoter activity, either by stimulating APP promoteractivity or retarding it.

[0100] The inhibition of APP promoter activity can, in turn, regulatethe expression of abnormal forms of tau. And, hence, the presentinvention also contemplates a method of regulating the expression ofabnormal forms of tau in a subject comprising modulating amyloidprecursor protein (APP) expression in the subject.

[0101] Moreover, compositions for modulating the expression, production,or formation of amyloid precursor protein (APP) in a subject areintended which comprise a carrier and cyclic adenosine monophosphate(cAMP), an analog of cAMP, a substance that is a ligand, an agonist, oran antagonist of a receptor coupled to the cellular levels of cAMP or toion channels, a compound that regulates the nuclear actions of cAMP orion channels, or a compound that regulates the activity of proteinkinase A.

5.2. Compositions of the Present Invention

[0102] As should be apparent, the present invention also contemplatescompositions comprising the active substances disclosed herein.Preferably, these compositions include pharmaceutical compositionscomprising a therapeutically effective amount of one or more of theactive compounds or substances along with a pharmaceutically acceptablecarrier.

[0103] As used herein, the term “pharmaceutically acceptable” carriermeans a non-toxic, inert solid, semi-solid liquid filler, diluent,encapsulating material, formulation auxiliary of any type, or simply asterile aqueous medium, such as saline. Some examples of the materialsthat can serve as pharmaceutically acceptable carriers are sugars, suchas lactose, glucose and sucrose, starches such as corn starch and potatostarch, cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt, gelatin, talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol, polyols such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters such as ethyl oleate and ethyl laurate, agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcoholand phosphate buffer solutions, as well as other non-toxic compatiblesubstances used in pharmaceutical formulations.

[0104] Wetting agents, emulsifiers and lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. Examples ofpharmaceutically acceptable antioxidants include, but are not limitedto, water soluble antioxidants such as ascorbic acid, cysteinehydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite,and the like; oil soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, aloha-tocopherol and the like; and the metalchelating agents such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

[0105] By a “therapeutically effective amount” or simply “effectiveamount” of an active compound, such as an analog of cAMP, is meant asufficient amount of the compound to treat or alleviate the negativeeffects of a neurological disorder or neurodegenerative disease stemmingfrom the aberrant expression, production, or formation of amyloidprecursor protein (APP) at a reasonable benefit/risk ratio applicable toany medical treatment. It will be understood, however, that the totaldaily usage of the active compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coinciding with the specificcompound employed; and like factors well known in the medical arts.

[0106] The total daily dose of the active compounds of the presentinvention administered to a subject in single or in divided doses can bein amounts, for example, from 0.01 to 25 mg/kg body weight or moreusually from 0.1 to 15 mg/kg body weight. Single dose compositions maycontain such amounts or submultiples thereof to make up the daily dose.In general, treatment regimens according to the present inventioncomprise administration to a human or other mammal in need of suchtreatment from about 1 mg to about 1000 mg of the active substance(s) ofthis invention per day in multiple doses or in a single dose of from 1mg, 5 mg, 10 mg, 100 mg, 500 mg or 1000 mg.

[0107] In certain situations, it may be important to maintain a fairlyhigh dose of the active agent in the blood stream of the patient,particularly early in the treatment. Hence, at least initially, it maybe important to keep the dose relatively high and/or at a substantiallyconstant level for a given period of time, preferably, at least aboutsix or more hours, more preferably, at least about twelve or more hoursand, most preferably, at least about twenty-four or more hours.

[0108] The compounds of the present invention may be administered aloneor in combination or in concurrent therapy with other agents whichaffect the central or peripheral nervous system, particularly selectedareas of the brain.

[0109] Liquid dosage forms for oral administration may includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs containing inert diluents commonly usedin the art, such as water, isotonic solutions, or saline. Suchcompositions may also comprise adjuvants, such as wetting agents;emulsifying and suspending agents; sweetening, flavoring and perfumingagents.

[0110] Injectable preparations, for example, sterile injectable aqueousor oleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

[0111] The injectable formulation can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions, which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

[0112] In order to prolong the effect of a drug, it is often desirableto slow the absorption of a drug from subcutaneous or intramuscularinjection. The most common way to accomplish this is to inject asuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug becomes dependent on therate of dissolution of the drug, which is, in turn, dependent on thephysical state of the drug, for example, the crystal size and thecrystalline form. Another approach to delaying absorption of a drug isto administer the drug as a solution or suspension in oil. Injectabledepot forms can also be made by forming microcapsule matrices of drugsand biodegradable polymers, such as polylactide-polyglycoside. Dependingon the ratio of drug to polymer and the composition of the polymer, therate of drug release can be controlled. Examples of other biodegradablepolymers include polyorthoesters and polyanhydrides. The depotinjectables can also be made by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissues.

[0113] Suppositories for rectal administration of the drug can beprepared by mixing the drug with a suitable nonirritating excipient,such as cocoa butter and polyethylene glycol which are solid at ordinarytemperature but liquid at the rectal temperature and will, therefore,melt in the rectum and release the drug.

[0114] Solid dosage forms for oral administration may include capsules,tablets, pills, powders, gelcaps and granules. In such solid dosageforms the active compound may be admixed with at least one inert diluentsuch as sucrose, lactose or starch. Such dosage forms may also comprise,as is normal practice, additional substances other than inert diluents,e.g., tableting lubricants and other tableting aids such as magnesiumstearate and microcrystalline cellulose. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings andother release-controlling coatings.

[0115] Solid compositions of a similar type may also be employed asfillers in soft-and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like.

[0116] The active compounds can also be in micro-encapsulated form withone or more excipients as noted above. The solid dosage forms oftablets, dragees, capsules, pills, and granules can be prepared withcoatings and shells such as enteric coatings and other coatings wellknown in the pharmaceutical formulating art. They may optionally containopacifying agents and can also be of a composition that they release theactive ingredient(s) only, or preferably, in a certain part of theintestinal tract, optionally in a delayed manner. Examples of embeddingcompositions which can be used include polymeric substances and waxes.

[0117] Dosage forms for topical or transdermal administration of acompound of this invention further include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants or patches. Theactive component is admixed under sterile conditions with apharmaceutically acceptable carrier and any needed preservatives orbuffers as may be required. Ophthalmic formulations, ear drops, eyeointments, powders and solutions are also contemplated as being withinthe scope of this invention.

[0118] The ointments, pastes, creams and gels may contain, in additionto an active compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

[0119] Powders and sprays can contain, in addition to the activecompounds of this invention, excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons.

[0120] Transdermal patches have the added advantage of providingcontrolled delivery of active compound to the body. Such dosage formscan be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the compound in apolymer matrix or gel.

[0121] Accordingly, the present invention is useful in the treatment oralleviation of disease, especially those disorders related toneurological diseases or neurodegenerative disorders, such asAlzheimer's disease, Parkinson's disease, Lou Gehrig's disease, ormultiple sclerosis, to name a few, not to mention central or peripheralnervous system damage, dysfunction, or complications involving samestemming from edema, injury, or trauma. Such damage, dysfunction, orcomplications may be characterized by an apparent neurological,neurodegenerative, physiological, psychological, or behavioralaberrations, the symptoms of which can be reduced by the administrationof an effective amount of the active compounds or substances of thepresent invention.

[0122] According to a specific embodiment of the present invention theadministration of effective amounts of anti-inflammatory agents cansuppress, inhibit, or neutralize the action of increased cAMP activity,which activity if unchecked leads to the overproduction of APP. Avariety of non-steroidal anti-inflammatory agents (NSAIDs) are found tobe suitable for reversing the stimulatory effects of cAMP, itsderivatives, a ligand, an agonist, or an antagonist of a receptor thatis coupled to the cellular levels of cAMP, or a compound that enhancesthe nuclear actions of cAMP. Examples of suitable NSAIDs include, butare not limited to, Advil, Aspirin, Aleve, Anaprox, Diclofenac,Docosahexaenoic acid, Dolobid, Etodolac, Feldene, Flurbiprofen,Ibuprofen, Indomethacin, Ketorolac tromethamine, Lodine, Meclofenamate,6-MNA, Motrin, Nalfon, Naprosyn, Nuprin, Orudis, Phenylbutazone,Piroxicam, Phenylbutazone, Ponstel, Relafen, Salicylic acid, Sulindacsulfide, Tolectin, Toradol, Voltaren; also 5-lipoxygenase inhibitors,phosphodiesterase inhibitors, or cyclooxygenase inhibitors (e.g.,cyclosalicylazosulfapyridine, azulfasalazine, DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),or DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone).

[0123] Other suitable anti-inflammatory agents may be salicylates, suchas Asacol, Disalcid, Pentesa, Salflex, or Trilisate; steroids and theircombinations, such as Aerobid, Aristocort, Azmacort, Beclovent,Beconase, Celestone, Cortenema, Cortifoam, Decadron, Delalone,Depo-Medrol, Dexacort, Epifoam, Hydeltra, Hydrocortone, Hydeltrasol,Medrol, Nasacort, Plaquenil, Pediapred, Rhinocort, Solu-Cortef,Vancenase, or Vanceril; receptor-site blockers, such as leukotriene, B₄,C₄, D₄ and E₄ receptor antagonists, prostanoid receptor antagonists,prostaglandin receptor antagonists, neurokinin receptor antagonists,endothelin receptor antagonists, antihistamines, cytokine/interleukinreceptor antagonists, or interferon receptor antagonist.

[0124] In a preferred embodiment of the invention the anti-inflammatoryagent is selected from the group consisting of steroidal ornon-steroidal anti-inflammatory agents, salicylates, steroids, receptorsite blockers, or inhibitors of complement activation.

[0125] In a preferred embodiment of the invention, the nonsteroidalanti-inflammatory agent is a specific inhibitor of cyclo-oxygenase type2, COX-2, activity, such as DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),resveratrol, or a mixture thereof.

[0126] Also according to the present invention, it is contemplated thatcertain immunotherapeutic treatments, especially those effective toinhibit an immune or inflammatory response, can be effective inreversing, inhibiting, or otherwise, alleviating the negative effects ofaberrant APP production. Such treatments include, but are not limitedto, the administration of immunoglobulins or other agents for theregulation of cytokine activity, for T- and B-cell activation, for Fcgreceptor blockade and for activating complement cascade; use ofrecombinant C3, CR1 and CR2 as inhibitors of serum complementactivation; immunotoxins, immunoligands or toxin fusion proteins; theadministration of vaccines or the oral administration of antigens.

[0127] Still other therapeutic “strategies” for preventing an immune orinflammatory reaction can be adopted including, but not limited to,cell/tissue transplantation, gene and stem cell therapy, adjuvanttherapy, extracorporeal therapy; use of telerogenic peptides,plasmapheresis and immunoadsorption.

[0128] Immune system suppressants or immunophilin ligands effective inthe methods of the present invention include, but are not limited to,immunomodulators, such as Ergamisol, Leukine, Neupogen,cyclophosphamide, colony-stimulating factors and the like, andimmunosuppressives, such as Atgam, Azathioprine, 15-Deoxyspergualin,HypRho, Imuran, Methotrexate, 6-Mercaptopurine, Mycophenolate mefotil(RS-61443), MICRhoGAM, Misoprostol, Methylprednisolone, Orthoclone,Prograf, Rapamycin, RhoGAM, Sandimmune, antithymocyte globulin (ATG),antilymphocyte globulin (ALG), monoclonal pan-T cell antibody (OKT3) andthe like.

[0129] Neurotransmitter antagonists or modulators of signal transductioncan be used to suppress the overproduction or overexpression of APP. Theaffected neurotransmitters include, but are not limited to adenosine,adrenoreceptors, angiotensin, atrial natriuretic peptide, bombesin,bradykinin, cholecystokinin, gastrin, dopamine, endothelin, GABA,glutamate, histamine, interleukin-1, serotonin, leukotriene, muscarinicacetylcholine, neuropeptide Y, nicotinic acetylcholine, opioid, PAF,prostanoid, purinoceptors, somatostatin, tachykinin, thrombin,vasopressin and oxytocin, VIP and the like.

[0130] Examples of modulators of kinase signaling (protein kinase A orC) include, but are not limited to phorbol esters, indolactam, mezerin,diacylglycerol, cAMP, CGMP, and their analogs; forskolin, activators orinhibitors of adenylate and guanylate cyclase; modulators of calcium orpotassium channels; G-proteins; and the like.

[0131] Examples of ion-channel modulators, e.g., antagonists, that canbe used according to the principles of the present invention includeN-acetylprocainamide HCl, amiloride HCl, 5-(N,N-dimethyl)-amiloride HCl,5-(N-ethyl-N-isopropyl)-amiloride, 5-(N,N-hexamethylene)-amiloride,5-(N-methyl-N-isobutyl)-amiloride, 4-aminopyridine, amiodarone HCl,apamin, R(+)-Bay K 8644, benzamil HCl, bepridil HCl, β-bungarotoxin,2,3-butanedione monoxime, calciseptine, charybdotoxin, μ-conotoxinGIIIA, ω-conotoxin GVIA, ω-conotoxin MVIIC, cyclic ADP ribose (cyclicADPR), cyclopiazonic acid, cyproheptadine HCl, dantrolene sodium salt,dendrotoxin, R(+)-DIOA, diltiazem HCl, efaroxan HCl, flunarizine HCl,fluspirilene, glibenclamide, glipizide, HA-1077 2HCl, 5-hydroxydecanoicacid sodium salt, IAA-94, iberiotoxin, kaliotoxin, lidocaine N-ethylbromide (QX-314), loperamide HCl, manoalide, MCD peptide, NAADP,nicardipine HCl, nifedipine, nifedipine metabolite, (±)-niguldipine HCl,S(+)-niguldipine HCl, R(−)-niguldipine HCl, nimodipine, nitrendipine,5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), phenamilmethanesulfonate, N-Phenylanthranilic acid, phentolamine mesylate,pimozide, procainamide HCl, quinidine sulfate, quinine sulfate,ruthenium red, ryanodine, saxitoxin, tetraethylammonium chloride,tetrodotoxin, tetrodotoxin citrate, thapsigargin, tityustoxin-Kα, TMB-8HCl, tolbutamide, triamterene, (±)-verapamil HCl, S(−)-verapamil HCl,R(+)-verapamil HCl, normethyl verapamil, (±)-methoxy-verapamil HCl,S(−)-methoxy-verapamil HCl, R(+)-methoxy-verapamil, and YS-035 HCl.These and related compounds can be obtained commercially, such as fromResearch Biochemicals International.

[0132] The following examples are provided for further illustration ofthe present invention, and do not limit the invention.

6. EXAMPLES

[0133] Experiments and exemplary procedures are described below whichprovide additional enabling support for the present invention. Inparticular, in vitro studies using primary cultures of rat corticalastrocytes and in vivo studies using appropriate animal models aredisclosed.

6.1. General Methods

[0134] Astrocytes are isolated from cortices from postnatal rats. See,K. D. McCarthy and J. de Vellis, J. Cell Biol. 85, 890 (1980). In brief,dissected cortices were dissociated by trypsinization and triturationthrough a flame-polished Pasteur pipette. Cells were plated ontopoly-L-lysine coated 35- or 100 mm culture dishes at densities of about10-25 cells/mm². The initial culture media, minimal essential medium(MEM, Gibco) containing 10% horse serum (BioWhittaker), were aspiratedafter 2-5 h after plating to remove unattached cells and debris, andreplaced with MEM containing 7.5% fetal bovine serum (FBS,BioWhittaker). Half the media was replaced with MEM/7.5% FBS twiceweekly. Cells are kept at 37° C. in a humidified 5% CO₂/95% airincubator. Media are changed twice weekly. Immunocytochemical stainingwith antibodies against GFAP and tau shows that >90% of cultured cellsare astrocytes and <5% are neurons. Pharmacological manipulations areperformed in serum-free media on 7-14 DIV confluent astrocytes.

[0135] 6.2. Detection of Cell-Associated Protein

[0136] To detect cell-associated protein (APP or GFAP), astrocytes from35 mm dishes are scraped in lysis buffer (60 mM Tris/HCl, 4% SDS, 20%glycerol, 1 mM dithiothreitol), ultrasonicated and boiled for 5 min. Thetotal amount of cell protein per dish, estimated using the bicinchoninicacid assay, is not altered by pharmacological treatments. Bromphenolblue (0.1%) is added to each sample and equal amounts of protein (−75mg/lane) are loaded on 10% SDS-polyacrylamide gels.

[0137] To detect secreted APP, culture media was collected after drugtreatments and phenylmethylsulfonyl fluoride was added to a finalconcentration of 2 mM. The media samples were then applied to SephadexPD-10 desalting columns (Pharmacia) and eluted with distilled water.Column eluates were frozen and dried by vacuum centrifugation. Thelyophilized proteins were reconstituted in 25 μL water followed by 25 μLof 2×Laemmli gel loading buffer, and boiled for 5 min.

[0138] The amount of media or cell protein loaded for sodium dodecylsulfate-polyacrylamide gel electrophoresis (10-20% SDS PAGE; Bio-Rad)was normalized for the amount of protein per sample. Proteins(equivalent to ˜100 μg cell protein/lane) were seperated byelectrophoresis, electroblotted onto polyvinylidene difluoride membranes(Immobilon-P, Millipore) and blocked in Tris-buffered saline with 0.15%Tween 20 (TBST) containing 5% powdered milk for 30 min. After 2×10 minrinses in TBST, the membranes were incubated in TBST containing anappropriate antibody. Monoclonal antibodies 22C11 and GFAP (both fromBoehringer-Mannheim) were used to detect the N-terminus of APP and glialfibrillary acidic protein respectively; antisera R37 and R98 (gifts ofDr. F. Kametani, Tokyo Institute of Psychiatry) were used to detectedthe C-terminus and KPI motifs of APP respectively; antiserum C8 (gift ofDr. D. Selkoe, Women's Hospital, Harvard Medical School, Cambridge, MA)was used to detect the C-terminus of APP.

[0139] After an overnight incubation, membranes were rinsed in TBSTbefore being treated for 1 h with a peroxidase-linked secondaryantibody. After several rinses in TBST, protein bands were visualized onKodak X-AR films by an enhanced chemiluminescence method (Amersham).Optical densities of the protein bands were quantitated by laserscanning densitometry (LKB, Bromma, Sweden), and normalized to thedensities of those bands generated under control conditions.

6.3. cAMP Assay

[0140] Levels of cyclic AMP were measured with [8-³H]-cAMP assay kit(Amersham TRK 432) in astrocytes grown on 35 mm dishes. In brief, afteraspirating the medium and rinsing twice with 1 ml ice cold PBS, thecells were scraped in 0.8 ml ice cold ethanol and sonicated. The cellsuspension was incubated for 5 min at room temperature, centrifuged andthe supernatant was dried in a rotary evaporator. After resuspension in120 μl Tris/EDTA buffer, two duplicate samples of 50 μl each were mixedwith the binding protein, [8-³H] adenosine 3′, 5′-cyclic phosphatetracer and incubated at 2-4° C. for 2 h. A charcoal suspension (100 μl)was added to the samples before centrifugation and 200 μl of thesupernatant were removed for scintillation counting. The amount ofcyclic AMP (pmol/mg protein) was estimated by comparing to knownstandards, and normalized to the amounts of whole cell protein asdetermined by the bicinchoninic acid assay (Sigma).

6.4. Data Analysis

[0141] Measurements of cellular and secreted proteins, or of mRNA intreatment groups were normalized against those of control groups whichwere prepared in parallel and loaded onto the same blot. Analysis ofvariance (ANOVA) and t-tests were used to evaluate differences betweengroups (significance level, p=0.05), using drug treatments as theindependent variable.

6.5. Analysis of RNA

[0142] Total RNA from astrocytes grown on 100 mm dishes is extracted bythe acid guanidium thiocyanate-phenol-chloroform method. See, P.Chomcznski and N. Saachi, Anal. Biochem. 162, 156 (1987). In brief, themedium was aspirated and the cells were scraped in 1 mL of TRI Reagent.After incubation for 15 min at room temperature, 0.2 ml chloroform wasadded, mixed vigorously with TRI Reagent and the mixture was stored foranother 15 min at room temperature. After centrifugation at 12,000 g for15 min, 0.5 ml isopropanol was added to the aqueous phase of the mixtureto precipitate RNA. The RNA pellet collected by centrifugation (12,000g, 15 min at 4° C.) was washed with 70% ethanol once and solubilized inan appropriate amount of Formazol (Molecular Research Center,Cincinnati, Ohio). RNA samples (˜20 μg) were denatured by heating for 15min at 60° C. prior to loading onto 1.2% agarose-formaldehyde gels forelectrophoresis. RNA was blotted onto Hybond polyvinyl membranes byovernight capillary transfer and fixed onto the membranes by UV lightillumination. Membranes were pre-hybridized with Amersham Rapid-hyb(Amersham Lab, Arlington Heights, Ill.) buffer for 2 h and labeledovernight with a ˜1.8 kb human APP cDNA (gift of Dr. Rachael Neve,McLean Hospital, Harvard Medical School, Belmont, Ma.) or humanglyceraldehyde-3-phosphate dehydrogenase probe (G3PDH; Clontech)labelled with [³²P]dCTP using random primed extension (AmershamMegaprime DNA labelling kit). Membranes were dried and exposed to KodakX-ray film for 24-48 h with an Amersham enhancer sheet. The relativeamounts of mRNA obtained by hybridization were estimated usingdensitometric analysis of autoradiographs. The levels of APP mRNA werenormalized to the amounts of G3PDH mRNA and expressed as a ratio to thelevels of untreated, control cells.

6.6. Exposure of Astrocytes to cAMP, NE and Others

[0143] Confluent monolayers of astrocytes prepared according to Example5.1 and treated with serum-free media containing 50, 100, or 250 μM8Br-cAMP for varying durations (1 h, 6 h, 12 h, or 24 h). Levels of cAMPin astrocytes are measured with a [8-³H] -cAMP assay kit (Amersham TRK432).

[0144] Cultures of astrocytes are also treated with norepinephrine (NE,50 and 100 μM) or with NE plus propranolol (50 μM). Cells are alsoexposed to NE (50 μM) or to the β-adrenergic agonist isoproterenol (50μM) to stimulate APP mRNA synthesis. The effects of other substances,including PMA, DNF, 8Br-cAMP, forskolin, prostaglandin E2, H8, H9,Win55212, Sp-cAMPS triethylamine and cyclosporin A are also observed,similarly.

[0145] Assays of PI hydrolysis are conducted as previously reported.Also, measurements of APPs are conducted as previously reported.

6.7. In Vivo Studies

[0146] The present studies indicate that immune system suppressants,such as cyclosporin A, can inhibit APP overexpression inGFAP-immunoreactive cultured astrocytes. Reactive astrocytes (that is,astrocytes that have been activated or stimulated in some fashion, e.g.,those associated with brain or neuronal injury) in vivo also upregulateGFAP expression. Indeed, the examination of post-mortem brains inpatients with Alzheimer's disease shows that reactive astrocytes arefound in proximity to amyloid plaques and regions of neurodegeneration.The inventors believe that neuronal, brain, or head injury gives rise tothe formation of reactive astrocytes, which overexpress APP andcontribute to the formation of amyloid or neurotoxic APP derivatives.

[0147] Thus, animal models of head injury and Alzheimer's diseaseexhibit increased amounts of APP in the brain. The in vivoadministration of immune system suppressants, like cyclosporin A, isfound to inhibit APP overexpression and the associated neurologicaldisorders. Inasmuch as head injury is associated with Alzheimer'sdisease (e.g., boxers with dementia pugilista), treatment withimmunosuppressants may prevent the progression of neuropathologicalsymptoms associated with this disease. Likewise, it is found that the invivo administration of agents that reduce or prevent astrocyticinflammation (e.g., reactive astrogliosis) reduces GFAP immunoreactivityand APP overexpression.

7. RESULTS

[0148] In summary, confluent primary rat cortical astrocytes, which aretreated with serum-free medium containing 50, 100, or 250 μM,respectively, of 8-Bromo-cAMP (8Br-cAMP) for 24 h, show a dose-dependentincrease in APP mRNA on Northern blots (120%, 150% and 180% of untreatedcells). In comparison, β-actin mRNA is decreased to 50% of untreatedcells by 8Br-cAMP (250 μM). Both L-norepinephrine (NE, 50 or 100 μM) andthe β-adrenergic agonist isoproterenol (50 μM) increase APP mRNA to 180%of untreated cells. The β-adrenergic antagonist propranolol (50 μM)reduces the NE-stimulated increase in APP mRNA to baseline levels. N-and C-terminal APP antibodies 22C11 and R37 (from Dr. F. Kametani, TokyoInst. Psychiatry), respectively, show that 8Br-cAMP or NE also increasesAPP holoprotein in cell lysates to 200% of that seen in untreated cells.Both drugs also increase process formation and glial fibrillary acidicprotein immunoreactivity in astrocytes. Thus, activation of β-adrenergicreceptors coupled to cAMP formation increases both APP mRNA andholoprotein in reactive astrocytes.

[0149] The APP gene promoter contains a consensus sequence for a cAMPresponse element (CRE). The immunosuppressant cyclosporin A (CycA, 10μM), which is known to inhibit CRE-mediated transcription, blocks theincrease in APP protein caused by 8Br-cAMP. This result suggests thatCycA may be used to prevent increases in APP and, potentially, amyloidformation in neurodegenerative diseases, such as Alzheimer's disease.

[0150] More specifically, and referring now to FIG. 1, confluentmonolayers of astrocytes are incubated for 24 h in serum-free mediacontaining 50, 100, or 250 μM 8Br-cAMP. A, Representative immunoblotwith mAb22C11 shows that astrocytes incubated with NE (50 μM) containsignificantly more cell-associated APP relative to untreated controlcells, Con (p<0.05), and that this increase is inhibited by theβ-adrenergic antagonist propranolol (Prop, 50 μM). This experiment isreplicated with similar results. B, Representative Northern blotindicates that two doses of NE (50 or 100 μM) are equally effective instimulating an increase in APP mRNA levels above those of controls(Con), and that this effect is mimicked by the β-adrenergic agonistisoproterenol (Iso, 50 μM). G3PDH mRNA is used as a control for RNAloading and is not affected by drug treatments. C, The stimulatoryeffect of NE or isoproterenol on APP mRNA synthesis (*, p<0.05) issignificantly inhibited by the β-adrenergic antagonist propranolol.

[0151] Referring now to FIG. 2, PI hydrolysis and cAMP formation aresignificantly increased by 1 h treatment with NE (50 μM). Co-incubationwith propranolol (50 μM) inhibits the increase in cAMP formation causedby NE (*, p<0.05) but has no effect on the increase in PI hydrolysis.Graphs represent means and SEM that are obtained from three independentexperiments.

[0152] Referring now to FIG. 3, A is a representative immunoblot thatshows that phorbol 12-myristate 13-acetate PMA (5 μM) ordexnorfenfluramine hydrochloride DNF (100 μM) treatment of astrocytesprepared according to Example 5.1 for 1 hour significantly increasesAPPs secretion relative to untreated cells (Con). B, indicates thatthese same drugs have no effect on the amount of cell-associated APP(APP holoprotein) after 24 hours. APP is detected by mAb22C11 on bothimmunoblots. Three experiments produce similar results.

[0153] Confluent astrocytes are prepared as in Example 5.1. The cellsare scraped and suspended in SDS-reducing sample buffer for loading onWestern blots. Referring now to FIG. 4, A is a representative immunoblotin which R37 antiserum and GFAP monoclonal antibody reveals two proteinbands at ˜120 kD (APP) and ˜50 kD (GFAP), respectively. B, charts theresults of 24 h exposure of the cells to increasing concentrations ofthe cAMP analog, 8Br-cAMP, or forskolin (50, 100 or 250 μM). As detectedby antiserum R37 or mAb22C11, the amounts of cell-associated APPincreases with increasing concentrations of both drugs (*, p<0.05). Thegraph presents data accumulated from 3 independent experiments.

[0154] Referring now to FIG. 5, APPs in the media is detected as a ˜110kD protein by mAb22C11. A, a representative immunoblot, shows thattreatment with 250 μM 8Br-cAMP for ca. 1 hour suppresses APPs secretion.Greater amounts of APPs accumulates in the medium after 24 hour exposureto 8Br-cAMP versus 1 hour. B, the graph shows that APPs is statisticallysignificantly reduced (*, p<0.05) after about 1 hour of exposure to8Br-cAMP, with the opposite trend after about 24 hour exposure. Thegraph represents the mean and SEM from three independent experiments.

[0155] Referring now to FIG. 6, astrocytes prepared as in Example 5.1are used. A representative Northern blot indicates an increase in APPmRNA with increasing concentrations of 8Br-cAMP. The G3PDH mRNA isunaffected by 8Br-cAMP treatments. Subsequent experiments show that 250μM is the most effective and reliable 8Br-cAMP concentration forstimulating APP synthesis.

[0156] Turning now to FIG. 7, the usual astrocytes are incubated with8Br-cAMP (250 μM) or NE (50 μM) for about 24 hours. A representativeNorthern blot shows that both drug treatments increase APP mRNA anddecrease β-actin mRNA levels. Three independent experiments producesimilar effects.

[0157] As before, astrocytes prepared according to Example 5.1 aretreated with increasing concentrations of 8Br-cAMP or forskolin for 24hours. As shown in the graph of FIG. 8, 8Br-cAMP and forskolin (both at50, 100, or 250 μM) cause significant increases in GFAP protein, asassayed using Western blots (*, p<0.05). The graph represents the meanand SEM obtained from 3 independent experiments.

[0158] In FIG. 9, A is a representative immunoblot showing that theincrease in cell-associated APP caused by 24 hour treatment with8Br-cAMP (250 μM) is suppressed by cotreatment with 1 μM cyclosporin A.This immunoblot also shows that cyclosporin A alone has no effect on thebasal APP levels. B, a representative immunoblot showing that basal APPssecretion levels are unaffected by 24 hour treatment with 8Br-cAMP, withor without 1 μM cyclosporin A. C, graphically shows how the suppressionof the 8Br-cAMP-induced increase in cell-associated APP is notsignificantly different at three doses of cyclosporin A (1, 5, or 10μM). The graph represents the mean and SEM of pooled data obtained from3 independent experiments.

[0159] It is also observed that the modulation of immune and/orinflammatory responses, which is achieved by the administration ofimmunosuppressants and/or anti-inflammatory agents, e.g., cyclosporin A,FK-506 and cannabinoid compounds; see, e.g., R. Condie, A. Herring, W.S. Koh, M. Lee, N. E. Kaminski, J. Biol. Chem., 271, 13175 (1996), inturn prevents or inhibits APP overexpression.

[0160] PG E₂ coupled to cAMP production increases the expression of APPholoprotein and mRNA.

[0161] Treatment of astrocytes for 24 h with 1, 10 or 100 μM PG E₂significantly increased the amounts of astrocytic APP mRNA (˜4.5 kb)relative to untreated cells (all p<0.05) (FIG. 10A). Similar increasesin APP holoprotein (˜110-130 kD) were detected by mAb 22C11, antiseraR37 or R98 on Western blots. Treatment with 1, 10 or 100 μM PG E₂produced increases in cellular APP holoprotein that were 1.4, 1.9 and2.3-fold respectively of untreated, control cells (FIG. 10B).

[0162] APP secreted in the media (˜110-130 kD) was also increased by 24h treatment with 1, 10 or 100 μM PG E₂ using mAb 22C11, antiserum R37 orC8 immunodetection (FIG. 10C). Treatment with 1, 10 or 100 μM PG E₂ alsostimulated dose-dependent increases in cellular cAMP levels to 27, 106and 227-fold compared to that of untreated cells (FIG. 10D); 0.1 μM PGE₂ did not stimulate cAMP production, and did not significantly alterAPP holoprotein or mRNA levels compared to untreated, control astrocytes(p>0.05).

[0163] Protein kinase A and cAMP regulate APP expression.

[0164] Treatment of astrocytes for 1 h with membrane-permeant 8Br-cAMP(250 μM) or by activating adenylate cyclase with forskolin (10, 50 or100 μM) significantly increased cellular cAMP levels (FIG. 11A), andsimilar increases in APP mRNA and holoprotein (FIG. 11B).

[0165] Activation of protein kinase A by 24 h treatment with 50, 100 or150 μM Sp-cAMP triethylamine increased cellular levels of APPholoprotein to 1.6, 1.9 and 2.2-fold compared to untreated cells (FIG.12A). By contrast, inhibition of protein kinase A with 100 μM of PKAinhibitor H-89 completely abolished the increase in APP holoproteinstimulated by 24 h treatment with 10 μM PG E₂ (FIG. 12B). APP wasdetected with antiserum R98 directed at the KPI motif of APP. Theseresults were replicated in subsequent experiments using mAb 22C11 or R37directed at the N-and C-termini of APP respectively.

[0166] Immunosuppressants cyclosporin A or FK-506 inhibit APP synthesisstimulated by PG E₂ or cAMP elevations. The increases in astrocytic APPholoprotein and mRNA stimulated by 24 h treatments with 50 M forskolinor 10 M PG E₂were significantly inhibited by co-treatment with either 50μM cyclosporin A or 50 μM FK-506 (FIG. 13). Representative Northern andWestern blots show that the increases in APP mRNA and APP holoprotein,but not the increases in GFAP levels, stimulated by PG E₂ (10 μM) areinhibited by cyclosporin A (CsA) or FK-506 (both 50 μM). Also, neithercyclosporin A nor FK-506 had a significant effect on cellular cAMPlevels stimulated by 10 μM PG E₂ (FIG. 14). Treatment of astrocytes witheither cyclosporin A or FK-506 (both 50 μM) alone had no significanteffect on basal APP holoprotein or CAMP levels (p>0.05).

[0167] Nicotinic ditartrate coupled to cAMP production increases theexpression of APP holoprotein.

[0168] Treatment of astrocytes for 24 h with 1, 10 or 100 μM nicotineditartrate significantly increased the amounts of astrocytic APPholoprotein relative to untreated cells (all p<0.05) (FIG. 15A). Similarincreases in APP holoprotein (˜110-130 kD) were detected by mAb 22C11,antisera R37 or R98 on Western blots.

[0169] APP secreted in the media (˜110-130 kD) was decreased by 1 htreatment with 1, 10 or 100 μM nicotine ditartrate using mAb 22C11,antiserum R37 or C8 immunodetection (data not shown).

[0170] Ion-channel modulator EGTA and calcium/calmodulin kinaseinhibitor KN-93 inhibit APP synthesis stimulated by PG E₂.

[0171] The increases in astrocytic APP holoprotein and mRNA stimulatedby 24 h treatments with 10 μM PG E₂ were significantly inhibited byco-treatment with either 100 μM EGTA (FIG. 15B) or 100 μM KN-93{N-[2-[[[3-(4′-chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4′-methoxy-benzenesulfonamidephosphate}, available from Research Biochemicals International (FIG.15C). Representative Northern and Western blots show that the increasesin APP mRNA and APP holoprotein, but not the increases in GFAP levels,stimulated by PG E₂ (10 μM) are inhibited by EGTA or KN-93 (both 100μM). Neither EGTA nor KN-93 had a significant effect on cellular cAMPlevels stimulated by 10 μM PG E₂ (data not shown). Treatment ofastrocytes with either EGTA or KN-93 (both 100 μM) alone had nosignificant effect on basal APP holoprotein or cAMP levels (p>0.05).

[0172] Effect of aspirin or resveratrol on APP expression and secretionin primary cultured astrocytes

[0173] Cultured astrocytes or neurons are treated with aspirin orresveratrol for 1 h (at nano- or micromolar range), and secretion ofsoluble APPs is measured by Western blot analysis. The increase in APPssecretion caused by aspirin or resveratrol is accompanied by decreasedlevels of cellular and amyloidogenic APP holoprotein (FIG. 16). Thus,NSAIDs appear to stimulate non-amyloidogenic APP processing in vitro.

[0174] Effect of DFU or DFP on APP expression and secretion in primarycultured brain cells.

[0175] Treatment of cultured astroyctes with DFU (A) or of culturedneurons with DFP (B) promotes APPs secretion and decreases the levels ofamyloidogenic APP holoprotein in cultured. Since COX-2 inhibitors mayoffer comparable efficacy without the risks of undesirable side-effectsassociated with non-specific COX inhibitors, we examine the effect ofspecific COX-2 inhibitors on APPs secretion and on the levels of APPholoprotein. FIG. 17 shows that treatment of cultured astrocytes withDFU or DFP (10⁻⁹−10⁻³M) for 1 h increases the secretion of soluble APPsand concurrently decreases the levels of cellular APP holoprotein. Thesedata indicate that specific COX-2 inhibitors can prevent the excessiveaccumulation of amyloidogenic APP, and also prevent the formation ofamyloid by enhancing the conversion of APP to form soluble APPs.

[0176] Aspirin inhibits APP overexpression caused by neuronal injury

[0177] Neuronal injury can stimulate prostaglandin production, andthereby stimulate APP overexpression. We injected rats withpara-chloroamphetamine (PCA), a neurotoxin which causes a loss ofserotoninergic terminals in the brain. We confirmed by microdialysisthat serotonin release in the striatum was compromised following chronictreatment with PCA, suggesting that serotoninergic terminals or synapsesmay be destroyed in rats injected with PCA.

[0178] Examination of brain tissue from the striatum or hippocampusrevealed that the levels of APP.holoprotein, as well as KPI-containingAPP isoforms, were increased by PCA injections (FIG. 18). In ratsinjected with both aspirin and PCA, the levels of APP holoprotein didnot differ from the levels of uninjected, control rats. Interestingly,injections of aspirin alone caused a significant decrease in the levelsof amyloidogenic APP holoprotein relative to control levels, an effectthat is consistent with our in vitro studies with cultures astrocytes orneurons. These data suggest that NSAIDs may be useful in preventing APPoverexpression in an in vivo model of AD.

8. CONCLUSION

[0179] Accordingly, the invention provides compositions and methods forpreventing, alleviating, or inhibiting abnormal APP synthesis by theadministration of antagonists (e.g., propranolol) of receptors that arecoupled to cAMP formation, particularly where the upregulation of suchreceptors and APP overexpression accompanies brain trauma, neurologicaldisease, or neurodegenerative disorder. The administration of suchreceptor antagonists suppresses cAMP formation, which in turn inhibitsabnormal APP synthesis.

[0180] Hence, the invention provides relief from the neuropathologicalsymptoms of diseases, such as Alzheimer's disease, by inhibitingaberrant APP gene expression. It is the overexpression of the APP genethat is believed to cause or strongly contribute to neurodegenerationand cognitive dysfunction in animals and humans.

[0181] It should be apparent to those of ordinary skill that thediscussion presented herein adequately supports the hypothesis that APPsynthesis (as evidenced by increases in mRNA and holoprotein) can beincreased by receptors coupled to a different messenger, cAMP formation.

[0182] It should also be apparent that other embodiments of theinvention can be readily contemplated by those of ordinary skill in theart after reviewing the present specification and teachings. The presentinvention is not limited, however, to the specific embodiments presentedherein and should not be construed so narrowly as to exclude embodimentsthat fall within the scope and spirit of the invention, which inventionis limited solely by the following claims.

What is claimed is:
 1. A method of modulating expression, production, orformation of amyloid precursor protein (APP) in a subject, comprisingadministering to said subject an effective amount of an antagonist of areceptor that is coupled to cellular levels of cAMP or to ion channels,wherein said antagonist is a non-steroidal anti-inflammatory agent thatis a specific inhibitor of cyclo-oxygenase activity, in apharmaceutically acceptable carrier.
 2. A method for enhancing secretionof a soluble form of APP in a subject, said method comprisingadministering to said subject a specific inhibitor of cyclo-oxygenaseactivity, in a pharmaceutically acceptable carrier.
 3. A method fordecreasing intracellular amyloidogenic APP holoprotein in a subject,said method comprising administering to said subject a specificinhibitor of cyclo-oxygenase activity, in a pharmaceutically acceptablecarrier.
 4. A method for decreasing overexpression of amyloidogenic APPand amyloidogenic APP peptides in a subject with a neurodegenerativecondition, said method comprising administering to said subject aspecific inhibitor of cyclo-oxygenase activity, in a pharmaceuticallyacceptable carrier.
 5. The method as in claim 4 wherein saidneurodegenerative condition is stroke, cerebral ischemia, ade-myelinating condition, or mechanical injury.
 6. A method for treatingimmune or inflammatory conditions associated with Alzheimers Disease ina subject, said method comprising administering to said subject aspecific inhibitor of cyclo-oxygenase activity, in a pharmaceuticallyacceptable carrier.
 7. A method for preventing Alzheimers Disease in asubject, said method comprising administering to said subject aneffective amount of a specific inhibitor of cyclo-oxygenase activity, ina pharmaceutically acceptable carrier.
 8. The method as in claim 1 inwhich said specific inhibitor of cyclo-oxygenase activity is a specificinhibitor of cyclo-oxygenase type 2 activity.
 9. The method as in claim2 in which said specific inhibitor of cyclo-oxygenase activity is aspecific inhibitor of cyclo-oxygenase type 2 activity.
 10. The method asin claim 3 in which said specific inhibitor of cyclo-oxygenase activityis a specific inhibitor of cyclo-oxygenase type 2 activity.
 11. Themethod as in claim 4 in which said specific inhibitor of cyclo-oxygenaseactivity is a specific inhibitor of cyclo-oxygenase type 2 activity. 12.The method as in claim 6 in which said specific inhibitor ofcyclo-oxygenase activity is a specific inhibitor of cyclo-oxygenase type2 activity.
 13. The method as in claim 7 in which said specificinhibitor of cyclo-oxygenase activity is a specific inhibitor ofcyclo-oxygenase type 2 activity.
 14. The method as in claim 8 in whichsaid specific inhibitor of cyclo-oxygenase type 2 activity is DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone), DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),resveratrol, or mixtures thereof.
 15. The method as in claim 9 in whichsaid specific inhibitor of cyclo-oxygenase type 2 activity is DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),resveratrol, or mixtures thereof.
 16. The method as in claim 10 in whichsaid specific inhibitor of cyclo-oxygenase type 2 activity is DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),resveratrol, or mixtures thereof.
 17. The method as in claim 11 in whichsaid specific inhibitor of cyclo-oxygenase type 2 activity is DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),resveratrol, or mixtures thereof.
 18. The method as in claim 12 in whichsaid specific inhibitor of cyclo-oxygenase type 2 activity is DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),resveratrol, or mixtures thereof.
 19. The method as in claim 13 in whichsaid specific inhibitor of cyclo-oxygenase type 2 activity is DFU(5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone),DFP(5,5-dimethyl-3-isopropyloxy-4-(4′-methylsulfonylphenyl)-2(5H)-furanone),resveratrol, or mixtures thereof.